1. Field of the Invention
This invention relates to supramolecules comprised of bioreactive conjugates, and to bioconjugates that bind to bioreactive molecules with a high affinity and regioselectivity. The invention also relates to arrays of bioreactive supramolecules formed from bioconjugates and to methods for their assembly,
2. Background of the Invention
Supramolecular chemistry or chemistry beyond the molecule, involves the arrangement of molecules bound via non-covalent interactions to form supramolecules (Lehn, Angew. Chem. Int. Edn. English 29:1304, 1990) Although the individual components are important for determining the specific nature of a supramolecular structure, the super structure allows for functionality not achievable with the isolated components. The classic supramolecules which emphasize this basic principle are nucleic acids and some polypeptides, which are endlessly produced by living organisms.
Methods for assembling supramolecular compounds are under investigation for use as detection systems and for new material properties (Vogtle, Supramolecular Chemistry, Wiley, Chichester, UK, 1991; Schneider et al., eds., Frontiers in Molecular Organic Chemistry and Photochemistry, VCH Publishers, Weinheim, Germany 1991). The development of supramolecular systems using combined functionalities of individual molecules is stimulated by natural models. Cells, as the ultimate molecular machines, use ensembles of proteins, nucleic acids and other macromolecules to perform complicated tasks such as replication, transcription and translation. Natural recognition systems such as receptor binding and nucleic acid hybridization enable biological systems to perform complex functions. One example of the unique functionality of supramolecules in nature is the machinery of photosynthesis. This complex of proteins and other biochemicals contains the supramolecular structure of the thylakoid membrane of chloroplasts (Bassi et al., Eur. J. Biochem. 204:317-26, 1991). Some of the better characterized multisubunit supramolecular biomolecules in nature are listed in Table 1.
TABLE 1Supramolecular Bioconjugates in NatureNo. ofOriginProtein ConjugateMol. Wt.SubunitsHumanα-Crystallin  810 kDa 30*PigLipoate Succinyl1,000 kDa 24*TransferaseCirraformiaErythrocruorin3,000 kDa162BovineDihydrolipoyl3,120 kDa 60TransacetylaseE. coliPyruvate Dehydrogenase4,600 kDa 24ComplexPhageFII Protein3,620 kDa180Cow Pea ChloroticMottle Virus4,608 kDa180ChickenAcetyl CoA4-10,000 kDa  CarboxylasePotato VirusX Protein35,000 kDa 650Tobacco Mosaic Virus40,000 kDa 2130 *= approximate
The highly specific functionality of single supramolecular biological compounds like E. coli pyruvate dehydrogenase complex, with 24 subunits and a molecular weight of 4 to 5 million is well recognized and utilized for numerous applications in biological and medical fields. However, little has been reported about synthetic combinations of naturally occurring molecules or their modified analogs to make supramolecular constructs with new and possibly enhanced, properties (Kabanov et al., Protein Engng. 4:1009-18, 1991).
The lack of uniform conjugation techniques and the long duration of current synthesis procedures serve as substantial barrels to regioselective supramolecular construction. Simple heterodimeric assemblies can be prepared by chemical cross linking (Wong, Chemistry of Protein Conjugation and Cross-linking, CRC Press, Boca Raton, Fla., 1991), but it is not always possible or economically feasible to cross-link the enormous variety of functional macromolecules available. Each macromolecule requires a special tailored procedure for cross linking. A uniform chemical conjugation procedure suitable for a wide variety of chemicals has not been developed. Furthermore, most bioreactive molecules have limited stability and would not endure long multiple step synthesis without losing their bioreactive properties. Limited chemical stability has discouraged the employment of successive conjugation strategies for the synthesis of supramolecular bioreactive constructs comprising labile moieties. The desire to limit the exposure of bioreactive subunits to inactivating conditions and to simplify or decrease the number of manipulative steps during supramolecular assembly have not been addressed by current technology.
Recombinant technology has been successfully employed for conjugating different regions of proteins not normally found adjacent in nature. While such technology is powerful, it is also limited to the biosynthetic capability of the host organism and subject to the limits of current technology. One limitation of recombinant DNA technology is the inability to synthesize non-proteinaceous compounds. In vivo expression place a weight limit of about 200 kilodaltons on recombinant proteins. Expression is limited to those proteins which are not toxic when expressed ill high levels in vivo. Proteins which are acidic, basic, hydrophobic or hydrophilic, may all be toxic and inexpressible at high levels.
There is a need for simplified methods for increasing both the regiospecificity and resolution, as well as reducing the cost of supramolecular compound construction. Improved methods which reduce cost, eliminate processing steps and minimize opportunities for inactivation and cross-contamination are highly desired but not addressed by current technology.